The objective is to isolate and characterize the locus of mating type determination in the unicellular eukaryote Tetrahymena thermophila. We propose to walk to the mat locus, starting from the rDNA gene located 30 centimorgans away on chromosome 2L; this distance is estimated to be in the order of 1-2 Mb. To this end, we will construct a library of inserts averaging 300 kb of Tetrahymena micronuclear DNA using a yeast artificial chromosome vector. Five to fifteen walking cycles should be sufficient to span the distance. Panels of Tetrahymena meiotic recombinant and vegetative asortant clones, used in conjunction with RFLPs, will allow us to determine a precise endpoint, to monitor progress, and to decide early on the correct direction of the walk. Once the mat locus has been reached, the DNA segments related to mating type determination will be identified by a variety of molecular and biological tests, and functionally characterized. The ultimate aim is a detailed genetic and molecular descritpion of the mechanism of mating type determination. Even before the mat locus is reached, the walk will yield valuable new knowledge on -- and molecular tools for the study of -- several fundamental aspects of MIC and MAC molecular genetics of Tetrahymena: - a measure of the kb of DNA corresponding to a centimorgan derived for meiotic mapping. - the molecular means to detect and measure the extent of genetic recombination within a MAC autonomously replicating piece (ARP). - a complete, ordered and mapped library of DNA inserts spanning an extended segment of MIC DNA. The genetic and molecular reagents made available by this walk will also be useful for other far-reaching investigations of the functional differentiation of MIC and MAC DNA (differences in DNA replication timing, in copy # control, in partition mechanism at nuclear division, and in transcriptional activity), as well as they study of the mechanism of this nuclear differentiation itself.